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Contents lists available at ScienceDirect
Molecular & Biochemical Parasitology
Evolution of the microtubular cytoskeleton (flagellar apparatus) in
parasitic protists
Naoji Yubuki a,b,∗ , Ivan Čepička b , Brian S. Leander a
a
The Departments of Botany and Zoology, Beaty Biodiversity Research Centre and Museum, University of British Columbia, 6270 University Blvd.,
Vancouver, BC V6T 1Z4, Canada
b
Department of Zoology, Faculty of Science, Charles University in Prague. Viničná 7, 128 44 Praha 2, Czech Republic
a r t i c l e
i n f o
Article history:
Received 24 October 2015
Received in revised form 2 February 2016
Accepted 5 February 2016
Available online xxx
Keywords:
Biodiversity
Cytoskeleton
Evolution
Free-living
Microtubules
MTOC
a b s t r a c t
The microtubular cytoskeleton of most single-celled eukaryotes radiates from an organizing center called
the flagellar apparatus, which is essential for locomotion, feeding and reproduction. The structure of the
flagellar apparatus tends to be conserved within diverse clades of eukaryotes, and modifications of this
overall structure distinguish different clades from each other. Understanding the unity and diversity of
the flagellar apparatus provides important insights into the evolutionary history of the eukaryotic cell.
Diversification of the flagellar apparatus is particularly apparent during the multiple independent transitions to parasitic lifestyles from free-living ancestors. However, our understanding of these evolutionary
transitions is hampered by the lack of detailed comparisons of the microtubular root systems in different
lineages of parasitic microbial eukaryotes and those of their closest free-living relatives. Here we help
to establish this comparative context by examining the unity and diversity of the flagellar apparatus in
six major clades containing both free-living lineages and endobiotic (parasitic and symbiotic) microbial
eukaryotes: stramenopiles (e.g., Phytophthora), fornicates (e.g., Giardia), parabasalids (e.g., Trichomonas),
preaxostylids (e.g., Monocercomonoides), kinetoplastids (e.g., Trypanosoma), and apicomplexans (e.g., Plasmodium). These comparisons enabled us to address some broader patterns associated with the evolution
of parasitism, including a general trend toward a more streamlined flagellar apparatus.
© 2016 Elsevier B.V. All rights reserved.
1. Introduction
The cytoskeleton of eukaryotic cells consists of an array of
microtubular and fibrous roots that stem from the basal bodies of
the flagellar apparatus [36]. The flagellar apparatus is a fundamental component of eukaryotic cells and is, therefore, among the only
ultrastructural systems that can be compared across the entire tree
of eukaryotes. The overall structure of the flagellar apparatus is relatively conserved but variable enough to identify homologous traits
between very distantly related lineages [36,68]. The high level of
conservation in the flagellar apparatus reflects its vital functions in
all eukaryotic cells, including division, shape, internal organization,
motility and feeding [35,53].
A comprehensive comparison of the flagellar apparatus across
the tree of eukaryotes has shown that the last eukaryotic common
∗ Corresponding author at: Department of Zoology, Faculty of Science, Charles University in Prague, Viničná 7, 128 44 Praha 2, Czech Republic. Fax: +420 22 195 1841.
E-mail addresses: [email protected] (N. Yubuki), [email protected]
(I. Čepička), [email protected] (B.S. Leander).
ancestor (LECA) already had a complex flagellar apparatus that was
most similar to the system found in free-living extant excavates
[68]. This suggests that the absence of specific traits, such as different flagellar roots, in many different groups of eukaryotes reflects
independent streamlining (i.e., trait losses) from a more complex ancestral condition. This punctate pattern of trait loss is also
observed within less inclusive taxonomic groups. For instance, the
flagellar apparatus in early branching prasinophycean green algae
(e.g., Pterosperma and Pyramimonas) is complex, whereas more
recently diverged core chlorophycean green algae (e.g., Chlamydomonas and Micromonas) have a much simpler flagellar apparatus
reflecting trait losses [33,52].
A large and diverse body of evidence supports the inference that
the LECA was a free-living, single-celled bacteriovore living in a
marine habitat. Free-living microbial lineages represent most of
eukaryotic diversity; endobiotic lineages (i.e., parasites, commensals and mutualists) are distributed in many different positions
across the tree of eukaryotes. Each major group of single-celled
eukaryotic parasites has emerged independently within a clade of
free-living lineages in order to exploit endobiotic lifestyles within
several different kinds of hosts (e.g., animals and plants). The tran-
http://dx.doi.org/10.1016/j.molbiopara.2016.02.002
0166-6851/© 2016 Elsevier B.V. All rights reserved.
Please cite this article in press as: N. Yubuki, et al., Evolution of the microtubular cytoskeleton (flagellar apparatus) in parasitic protists,
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Fig. 1. Illustration showing the general molecular phylogenetic relationship of the specific lineages of eukaryotes addressed in this review. Lineages of mostly free-living
eukaryotes that are not relevant to this review have been trimmed for clarity (e.g., ciliates, dinoflagellates, brown algae, euglenids). The names in bold denote the major
groups of eukaryotes.
sition to a parasitic lifestyle from free-living ancestors involved
substantial changes at the genomic, ultrastructural and behavioral
levels. In this review, we examine the evolutionary diversification of the microtubular root systems of flagellar apparatus in six
different groups of parasitic protists within the context of their
nearest free-living relatives: stramenopiles, fornicates, kinetoplastids, parabasalids, preaxostylids and apicomplexans (Figs. 1 and 2).
These six lineages are nested within different eukaryotic supergroups. Stramenoples and apicomplexans (Myzozoa, Alveolata)
both fall within a more inclusive group called SAR (Stramenopiles,
Alveolata and Rhizaria), which consists of many different lineages
with diverse in morphologies; SAR has been established on the
basis of molecular phylogenetic analyses rather than shared morphological features [13]. Fornicates, kinetoplastids, parabasalids
and preaxostylids are nested within the Excavata, which also
includes photosynthetic lineages (e.g., Euglena) and free-living heterotrophic flagellates (e.g., Jakoba). The best synapomorphy for
excavates is a conspicuous feeding groove on the ventral side of
the cell, which is supported by several microtubular roots stemming from the flagellar apparatus [58]. Broad-level comparisons of
the flagellar apparatus across the tree of eukaryotes enabled us to
identify some reoccurring trends associated with the independent
evolution of parasitic lineages in microbial eukaryotes. The focus
on microtubules more than fibrous structures is intentional. While
fibers in the flagellar apparatus undoubtedly play an important role
as a cellular component in eukaryotes, their homology across different lineages is usually elusive because of diverse morphologies.
Comparisons of these ultrastructural data are also affected by the
application of different methodologies, such as fixation and staining
protocols.
2. The flagellar apparatus of parasitic protists
Biologists working within the context of different groups of
parasites have inadvertently applied different terms for homologous traits, making comparisons of cytoskeletal systems in different
groups of parasites unnecessarily difficult. In order to avoid con-
fusion when comparing homologous traits associated with the
flagellar apparatus, we will use terminology that can be applied
to all eukaryotes as advocated previously [36,68].
The flagellar apparatus in most microbial eukaryotes usually
consists of two basal bodies that anchor the flagellar axonemes
and a system of microtubular and fiberous roots (Fig. 3). Each basal
body is usually associated with two different microtubular roots,
producing a total of four roots within the cell. The oldest (most
posterior) basal body is labeled “1” (i.e., basal body 1 or B1), and
the youngest (most anterior) basal body is labeled “2” (i.e., basal
body 2 or B2). The two microtubular roots associated with basal
body 1 are labeled “root 1” (i.e., R1) and “root 2” (i.e., R2); the two
microtubular roots associated with basal body 2 are labeled “root
3” (i.e., R3) and “root 4” (i.e., R4) in a clockwise orientation when
viewed from the tip of the flagellum [36]. A single microtubular
root (i.e., SR) also originates from B1 and is located between R1 and
R2. This SR is well characterized in excavates, ancyromonads and
apusomonads and has also been observed in some of the earliest
diverging lineages of stramenopiles [23,69]. The SR consists of only
a single microtubule and is, therefore, easily overlooked, so the true
distribution of this root might be much broader than the currently
available descriptions indicate.
3. The Stramenopiles
The Stramenopiles is a diverse clade of mostly microbial
eukaryotes living in marine, freshwater, terrestrial, and endobiotic
environments [39]. Trophic modes within the group include photoautotrophy (e.g., brown algae and diatoms), phagotrophy (e.g.,
bicosoecids Cafeteria), saprotrophy (e.g., Labyrinthulomycetes),
and parasitism (e.g., Oomycetes and Blastocystis). Molecular phylogenetic evidence demonstrates that the most recent ancestor
of stramenopiles was likely a free-living bacterivorous biflagellate [55]. Parasitic stramenopiles have a polyphyletic distribution
within the group and therefore evolved independently several
times [14]. Oomycetes (or Peronosporomycetes), for instance,
are fungus-like rhizoidal organisms with a flagellated zoospore
Please cite this article in press as: N. Yubuki, et al., Evolution of the microtubular cytoskeleton (flagellar apparatus) in parasitic protists,
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Fig. 2. Light micrographs representing the major eukaryotic groups including both free-living and parasitic lineages. All scale bars are 10 ␮m except for 100 ␮m in (c) and (l).
(a) Stramenopile, Bicosoeca (Bicosoecida); (b) Stramenopile, Ulkenia (Labyrinthulomycetes), taken with permission from Ref. [66]; (c) Stramenopile, Cepedea (Slopalinida);
(d) Myzozoa, Chromera (Chromerida); (e) Myzozoa, Colpodella (Colpodellida), taken with permission from Ref. [32]; (f) Myzozoa, Plasmodium (arrowheads) (Apicomplexa);
(g) Fornicata, Kipferlia (CLO); (h) Fornicata, Chilomastix (Retortamonadida); (i) Fornicata, Giardia (Diplomonadida); (j) Parabasalia, Pseudotrichomonas (Trichomonadida);
(k) Parabasalia, Tetratrichomonas (Trichomonadida); (l) Parabasalia, Trichonympha (Trichonymphida); (m) Preaxostyla, Trimastix (Trimastigida); (n) Preaxostyla, Monocercomonoides (Oxymonadida); (o) Preaxostyla, Pyrsonympha (Oxymonadida); (p) Kinetoplastida, Bodo (Eubodonida); (q) Kinetoplastida, Trypanoplasma (Parabodonida). (r)
Kinetoplastida, Trypanosoma (Trypanosomatida).
stage (e.g., the infamous potato pathogen Phytophthora infestans).
Blastocystis is an anaerobic parasite that inhabits the lower gastrointestinal tract of humans and a wide range of other animals;
it is the only stramenopile known to commonly infect humans
[54]. Molecular phylogenetic analyses show that Blastocystis is
closely related to slopalinids, which are anaerobic intestinal commensals of animals, especially anuran amphibians. Slopalinids are
either small biflagellated cells (around 15 ␮m) with one nucleus or
large multiflagellated cells (up to several millimeter) with multiple nuclei. Blastocystis plus the slopalinids form one of the earliest
branching lineages of the stramenopiles along with the freeliving Bicosoecida, the free-living Placididea, and the saprotrophic
Labyrinthulomycetes [14]; the branching order among those lineages still remains unclear.
The flagellar apparatus is relatively uniform within members
of the Stramenopiles, despite the very high level of diversity
in morphology, behavior and modes of nutrition, except for the
modifications found in slopalinids and Blastocystis. Stramenopiles
typically possess four microtubular roots that radiate from two
basal bodies. The free-living Cafeteria (Bicosoecida), however, has
two roots, R1 and R2, associated with the posterior B1 and only
one root, R3, associated with the anterior B2; this lineage has lost
R4. Cytoplasmic microtubules extend from R3 to support the dorsal
side of the cell (Fig. 4a). R2 divides into two subroots that reinforce
a feeding pocket used in bacterivory [45]. The flagellar apparatus in
the zoospore of the parasitic Phytophthora (Fig. 4b) is very similar to
the flagellar apparatus in Cafeteria, the zoospores of labyrinthulids
(e.g., Thraustochytrium) and most photosynthetic stramenopiles.
R1 and R2 originate from the posterior B1 and extend posteriorly to the left and right sides of a ventral furrow, respectively
[1]. The zoospores of Thraustochytrium do not perform phagotrophy and do not have a cytostome; however, the zoospores still
possess an R2 that is divided into two subroots [1]. An anteriorly
directed R3 from the anterior B2 supports an array of cytoskeletal
microtubules, which is a common configuration in stramenopiles.
In slopalinids, Proteromonas with two flagella and Karotomorpha
with four flagella represent the early branching lineages in this
group; members with multiple flagella and multiple nuclei, such as
Opalina and Protoopalina, are more recently derived [28]. Slopalinids have lost the basic flagellar root system and the cytostome found
in free-living lineages of stramenopiles. The cone shaped anterior
end of Proteromonas is supported by microtubules extending from
two anterior basal bodies [9] (Fig. 4c); the folded cell surface of
Protoopalina is supported by rows of basal bodies and fibrous connectives) [46]; Blastocystis has completely lost basal bodies and a
microtubular cytoskeleton beneath the cell surface.
4. The Myzozoa (Alveolata)
The Myzozoa consists of two major subgroups – dinoflagellates and apicomplexans – plus several other lineages of parasites,
symbionts and eukaryovorous flagellates (e.g., Perkinsus, Chromera
and Colpodella). Apicomplexans are obligate intracellular parasites,
some of which cause major diseases in livestock and humans
world-wide. For example, Plasmodium spp. (the causative agent of
malaria), Cryptosporidium spp. and Toxoplasma gondii are significant
human pathogens. The feature that distinguishes this group from
other eukaryotes is the “apical complex”, which functions in host
cell invasion. The apical complex contains structural elements, such
as a polar ring that organizes the subpellicular microtubules, and
secretory organelles, called rhoptries and micronemes, that release
enzymes during the cell invasion process. The closest free-living
relatives of the obligately parasitic apicomplexans are photosynthetic symbionts of corals (e.g., Chromera) and predators (e.g.,
Colpodella) [29,37,43]. Chromera possesses a functional chloroplast.
Colpodella is a predatory biflagellate that feeds by sucking out the
cell contents from prey cells (i.e., myzocytosis) with a cytoskeletal/secretory apparatus that is homologous to the apical complex
in apicomplexans [31,59].
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Fig. 3. Transmission electron micrographs showing the basal bodies, major microtubular roots and fibers of the flagellar apparatus in representative lineages of eukaryotes.
Scale bars, 500 nm. (a) The Stramenopiles, Apoikia (Chrysophyta). (b) The Fornicata, Kipferlia (CLO). (c) The Parabasalia, Pseudotrichomonas (Trichomonadida). HL, SF and P
refer “hooked lamina”, “sigmoidal fiber” and “pelta” microtubular rows, respectively. (d) The Preaxostyla, Monocercomonoides (Oxymonadida). Pax refers to “preaxostyle”.
The diversity and evolution of the cytoskeleton in apicomplexans and their free-living relatives was reviewed recently [43,44].
Except from some microgametes, apicomplexans have essentially
lost the flagellar root system altogether [40]. The ultrastructure of
microgametes in Toxoplasma shows two possible root structures:
(1) a posterior root with five microtubules extending from the
area between two basal bodies and (2) a row of 15 short microtubules positioned in the anterior end of the cell [47]. The position
of the posterior root with five microtubules is most similar to R1 in
Colpodella and other myzozoan flagellates. There are three microtubular roots in Colpodella: a “bypassing” microtubular root, R1
from the posterior B1, and R4 from the anterior B2. The bypassing root connects with the dorsal side of B1. Although this root
extends toward the anterior end of the cell, it does not merge with
the apical complex. R1 is relatively small and short, and R4 extends
to the apical complex [7]. The flagellar apparatuses of Chromera
and Colpodella are similar, but Chromera has a short R2 positioned
between the two basal bodies. The bypassing root in Chromera terminates at the anterior end of the apical complex [50].
Apicomplexans also have a fiber that connects the apical complex to the centrioles and consists of “striated fiber assembly” (SFA)
proteins that are essential for cytokinesis and the inheritance of
cytoskeletal structures into the daughter cells [20]. SFA proteins
were originally described in green algae (i.e., Chlamydomonas) as an
organizing element connecting the flagellar roots to the basal bodies [73]. However, because apicomplexans with the exception of
the microgamates have lost the flagellar apparatus altogether [20],
speculated that the ancestral apicomplexan retained the SFA fiber
from their free-living biflagellated relatives to organize cell division. Because homologous SFA proteins have been detected in two
very distantly related lineages of eukaryotes (i.e., green algae and
apicomplexans), these proteins were either inherited from their
most recent common ancestor or acquired by a more recent lateral
gene transfer event. The SFA fiber in apicomplexans is probably
homologous with R4 in Colpodella and Chromera, so it would be
interesting to demonstrate whether R4 and any associated fibers in
these lineages contain SFA proteins.
5. The Fornicata (Excavata)
The Fornicata represents one of the major lineages within the
much more inclusive Excavata. Fornicates are organized within
three assemblages: Carpediemonas-like organisms (CLOs), the
Retortamonadida (e.g., Retortamonas) and the Diplomonadida (e.g.,
Giardia). CLOs represent a paraphyletic group of free-living flagellates that encompasses the most recent ancestor of all fornicates.
Retortamonads also represent a paraphyletic/polyphyletic group
within fornicates [15,27,63]. Except for one free-living species (i.e.,
Chilomastix cuspidata), retortamonads are either obligate commensals or parasites within the guts of animals. Some members of
the Diplomonadida are the causative agents of important diseases
such as the human parasite Giardia intestinalis and the fish parasites Spironucleus spp.; some diplomonads, however, have become
free-living organisms presumably from parasitic ancestors (e.g.,
Trepomonas agilis) [56]. Members of the Diplomonadida have either
a single flagellar apparatus and an associated nucleus (single karyomastigont) or, more often, two flagellar apparatuses (usually
with eight flagella in total) and two associated nuclei (double
karyomastigont). Because both single- and double-karyomastigont
organisms are paraphyletic/polyphyletic groups in molecular phylogenetic analyses, the evolution of the karyomastigont cell system
is still unresolved [26].
The free-living CLOs have a typical excavate flagellar apparatus with two flagella, which is one of the most complex flagellar
apparatuses known and is inferred to approximate the flagellar
apparatus in the LECA [68]. The main microtubular roots originate
from the posterior B1 and reinforce the ventral feeding groove:
R1, R2 and SR (i.e., the singlet root) (Fig. 4d). The cytopharynx
at the base of the ventral groove is mainly supported by split R2
roots. The anterior B2 anchors a relatively small R3 that extends
to the anterior part of the cell. In retortamonads, four flagella are
grouped into two pairs, but the flagellar root system is very similar
to the one of free-living CLOs described above [3,6]. The cytoskeletal
structure in retortamonads also includes the interlinked microtubular corset positioned underneath the cell membrane [5]. The
flagellar apparatus of diplomonads is much simpler and contains a
smaller microtubular root system than those in CLOs and retortamonads. R1, R2 and R3 have traditionally been referred to as
the “infranuclear fiber” (Inf), “cytostomal fiber (or funis)” (Cf), and
“supranuclear fiber” (Snf), respectively. In diplomonads wherein
the flagellar apparatus has doubled to form two “kinetids” (e.g.,
Hexamita), the R1’s of each kinetid cross in the middle of the cell
and the R3’s of each kinetid cross in the anterior region of the cell
(Fig. 4e) [11]. The evolution of the flagellar apparatus in the Fornicata appears to be associated with the evolution of a feeding groove
on the ventral side of the cell; prominent microtubular roots, R1
and R2 are associated with the large feeding grooves in CLOs and
retortamonads, and simpler roots are associated with either feeding pockets (e.g., Trepomonas) or feeding tubes (e.g. Hexamita) or
the absence of a feeding structure (e.g., Giardia) in diplomonads.
Giardia intestinalis is a parasitic diplomonad that causes intestinal disease in humans. Giardia attaches to the epithelium of the
small intestine using a specialized microtubule structure called a
“ventral disc” (Fig. 3f). This adhesive disc is supported by a spiral
array of parallel microtubules (vdMts) associated with the “median
body” (MB), the “funis” and the “supernumerary microtubule array”
(snMTs) [18,24] (Fig. 3f). The MB is a semi-organized microtubule
array that is positioned on the ventral side of the cells. Although its
function is unknown, researches have suggested several different
possibilities such as the storage of prepolymerized tubulin for the
ventral disc assembly for the next generation or participation in
caudal tail flexion [48]. The funis is a sheet of microtubules asso-
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Parasitic or commensal
Free-living
(a)
(b)
R3
(c)
R4
Stramenopiles
2
2
1
2
1
R3
1
R1
R2
R2
R1
Cafeteria
(d)
Phytophthora
(e)
R3
Proteromonas
(f)
vdMts (R3?)
Snf (R3)
Fornicata
snMts
Inf (R1)
funis (R2?)
R2
SR
R1
MB
Cf (R2)
Kipferlia
Hexamita
Giardia
Fig. 4. Illustration of the flagellar apparatus of Stramenopiles (a–c) and Fornicata (d–f). The flagellar roots in the same colors correspond each other. Arrows indicate the
direction of flagella. (a) Free-living Cafeteria roenbergensis (Bicosoecida), redrawn based on the data in Ref. [45]. (b) Parasitic Phytophthora parasitica (oomycetes), redrawn
based on the data in Ref. [1]. (c) Endobiotic Proteromonas lacertae (Slopalinida), redrawn based on the data in Ref. [9]. (d) Free-living Kipferlia bialata (‘Carpediemonas-like
organism’, CLO), redrawn based on the data in Ref. [71]. (e) Parasitic Hexamita (Hexamitinae, Diplomonadida), redrawn based on the data in Ref. [6]. The Inf, Cf and Snf refer
to infranuclear fiber, cytostomal fiber, and supranuclear fiber, respectively. (f) Parasitic Giardia (Diplomonadida), redrawn based on the data in Refs. [2,12]. The vdMTs, snMTs
and MB refer to ventral disc microtubule array, supernumerary microtubule array and median body, respectively.
ciated with a caudal axoneme and is located on the posterior side
of the cell. The snMTs form a partial, left-handed, spiral array that
is positioned against the microtubular array that forms the ventral
disc (vdMts). The relationships of these roots to each other and the
basal bodies suggest that the funis and the vdMts are homologous
with R2 and R3, respectively, in other diplomonads (and eukaryotes
as a whole) (Fig. 4f).
6. The Parabasalia (Excavata)
The Parabasalia contains a morphologically diverse assemblage
of mostly parasites/commensals of insects and vertebrates; for
instance, trichomonads are relatively small cells (∼10–20 ␮m)
with six or fewer flagella and hypermastigids are very large cells
(∼200 ␮m) with up to thousands of flagella [11]. Hypermastigids
play an important role in the digestion of cellulose within the
guts of termites and wood eating cockroaches. Some trichomonads are important parasites of animals, including humans (e.g.,
Trichomonas vaginalis and Tritrichomonas foetus). The Parabasalia
contains very few free-living representatives, and these lineages,
have a punctate distribution among parasitic species in molecular phylogenetic analyses [70]; this suggests that the free-living
lineages of trichomonads evolved from parasitic ancestors several
times independently. Moreover, molecular phylogenetic data also
suggest that the hypermastigid cell type evolved multiple times
independently from simpler ancestors. Therefore, “trichomonads”
and “hypermastigids” represent morphotypes within parabasalids
rather than clades [16,41].
The Parabasalia are distinguished from other eukaryotes by the
presence of hydrogenosomes, and a “parabasal apparatus” consisting of striated fibers connecting the Golgi body to the flagellar
apparatus and the absence of a cytostome. Although some species
are aflagellated (e.g., Dientamoeba) and some are multiflagellated,
the typical arrangement of the parabasalid flagellar apparatus consists of four basal bodies: one directed posteriorly (B1) and three
directed anteriorly (B2–B4). The anteriorly directed basal bodies
have distinctive fibrous appendages: B2 has a sigmoidal fiber; B3
and B4 have a hooked lamina (Fig. 3b). The primary cytoskeletal feature composed of microtubules, called the “pelta-axostyle
complex”, has been inferred to be homologous with the peripheral
microtubules of other eukaryotic groups [58]. The pelta is a microtubular array that covers the anterior part of the cell. The axostyle
is a sheet of microtubules arranged as either a hollow tube or a cone
and reinforces the longitudinal axis of the cell. Many trichomonads
have a conspicuous striated fiber, called the “costa” that extends
posteriorly from the region containing the basal bodies. The costa is
inferred to be homologous with the “C fiber” associated with R1. The
C fiber is a thick structure with a multilayered appearance divided
by distinct sheets and is commonly found in other excavates, such
as jakobids and the free-living Kipferlia [60,71]. As reviewed elsewhere, various modifications of this basic cytoskeletal arrangement
are found in different lineages of parabasalians [8,11,16].
7. The Preaxostyla (Excavata)
The Preaxostyla is a relatively small group of excavates that
contains two subgroups of heterotrophic flagellates living in low
oxygen environments: the Oxymonadida and the Trimastigida [58].
The Oxymonadida is a group of either obligate symbionts that live
mostly in the hindguts of termites and wood eating cockroaches.
Members include the streamlined and relatively tiny (ca. 10 ␮m)
Monocercomonoides with four flagella and the relatively large (ca.
200 ␮m) and multinucleated Barroella with numerous axostyles
throughout the cell body. Oxymonads are among the most problematic and interesting groups of microbial eukaryotes from an
evolutionary perspective. Oxymonads are nested within a paraphyletic assemblage of free-living trimastigids, which brings the
Preaxostyla firmly within the Excavata [72].
The free-living Trimastix has four basal bodies associated with
four flagella. Trimastigids have most of the cytoskeletal traits
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Parasitic or commensal
Free-living
R3
(a)
(b)
Pelta
(c)
2
3
2
3
2
Preaxostyla
3
R2
(Pax)
4
1
1
R2
SR
4
R1
1
4
Axostyle
SR
I fiber
Oxymonas
Monocercomonoides
Trimastix
(d)
Kinetoplastea
R2
(Pax)
R3
R1
(e)
(f)
R3
R3
R2
(MTR)
R2
(MTR)
2
MTQ (R3)
R1
1
2
R1
1
2
1
Bodo
Cryptobia
Trypanosoma
Fig. 5. Illustration of the flagellar apparatus of Preaxostyla (a–c) and Kinetoplastea (d–f). The flagellar roots in the same colors correspond each other. Arrows indicate the
direction of flagella. The number of microtubule shown in each cross section for microtubular corset is not significant here. (a) Free-living Trimastix marina (Trimastigida),
redrawn based on the data in Ref. [62]. (b) Commensal Monocercomonoides hausmanni (Oxymonadida), redrawn based on the data in Refs. [51,62,58]. The Pax refers to
preaxostyle. (c) Parasitic Oxymonas (Oxymonadida), redrawn based on the data in Ref. [10] (d) Free-living Bodo spp. (Eubodonida), redrawn based on the data in Refs. [4,55].
The MTR refers to the reinforced microtubular band. (e) Parasitic Cryptobia iubilans (Parabodonida), redrawn based on the data in Ref. [42]. (f) Parasitic Trypanosoma brucei
(Trypanosomatida), redrawn based on the data in Refs. [19,30]. The MTQ refers to the microtubule quartet.
associated with the typical excavate flagellar apparatus like that
found in Kipferlia and retortamonads (Fornicata): three microtubular roots (R1, R2 and SR) from the posterior B1 and an R3 from the
anterior B2 (Fig. 5a). The cytopharynx at the base of the ventral
groove in Trimastix is mainly supported by two bands of microtubules stemmed from R2. The ventral (or inner) face of R2 near
the anterior end of the cell is associated with lattice-like “I fiber”
[58].
Oxymonads have a distinctive cytoskeletal organization composed of four flagella organized as two pairs of basal bodies
separated by a striated structure called the “preaxostyle”. Oxymonads (e.g., Pyrsonympha) also have a dynamic “axostyle” composed
of an array of parallel and interlinked microtubules that run
along the longitudinal axis of the cell. An anterior array of microtubules, called the “pelta”, supports the anterior part of cell.
Detailed examination of the flagellar apparatus in the oxymonad
Monocercomonoides hausmanni confirmed that the cytoskeletal
organization is very similar to Trimastix and other excavates [51,62].
The most posterior basal body (B1) is associated with two microtubular roots: R1 runs posteriorly beneath the posterior flagella
(Fig. 5b). Simpson et al. [62] suggested that the preaxostyle complex in Monocercomonoides and other oxymonads is homologous to
R2 and the “I fiber” in Trimastix. The most anterior basal body (previously called B4) is actually B2 and is associated with the posteriorly
directed R3 (previously called R2) from which the microtubules of
the pelta extend [58].
Some termite symbionts (e.g., Oxymonas and Pyrsonympha)
attach to the intestinal wall of their hosts by a holdfast structure
consisting of “microfibrils”. Oxymonas, for instance, has a long anterior “rostellum” that is supported by microtubules and terminates
in the holdfast [10]. This ribbon of microtubules runs though the
entire cell body from the anterior holdfast to the posterior end
of the cell. The flagellar apparatus of Oxymonas is therefore very
divergent; the only remnant of the excavate configuration of microtubular roots in this genus is a potential homolog of R2, namely the
preaxostyle that separates the two pairs of basal bodies (Fig. 4c).
8. The Kinetoplastea (Excavata)
The Kinetoplastea is one of four major subgroups within the
Euglenozoa (Excavata), all of which share three distinguishing
cytoskeletal traits: paraflagellar rods within the flagella, tubular extrusomes and a conserved flagellar root system [57]. Most
lineages in the Euglenozoa are free-living (e.g., euglenids, symbiontids and diplonemids), but parasitic lifestyles evolved several
times independently within the Kinetoplastea [34,61]. A shared
feature of kinetoplastids is a distinctive mitochondrial inclusion
called a “kinetoplast”, which is a condensed mass of DNA (i.e.,
kDNA). Although many lineages of kinetoplastids are parasites of
animals, the most recent ancestor of all kinetoplastids was clearly
free-living, like the vast majority of other euglenozoans. However,
prokinetoplastids infect a wide range of marine and freshwater fish
and represent some of the earliest diverging lineages within the
Kinetoplastea in molecular phylogenetic trees. The Eubodonida,
Neobodonida and Parabodonida are mainly free-living flagellates
that play important roles in aquatic ecosystems as consumers
of bacteria and detritus. Many members of Bodo are common,
abundant and cosmopolitan. A few lineages of bodonids, such as
Cryptobia and Trypanoplasma, parasitize commercially important
fish [65]. Trypanosomatids, by contrast, are obligate parasites of
Please cite this article in press as: N. Yubuki, et al., Evolution of the microtubular cytoskeleton (flagellar apparatus) in parasitic protists,
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animals, including humans. Trypanosoma brucei, for example, is
among the best-studied kinetoplastids because it is the causative
agent of African sleeping sickness.
The general arrangement of the flagellar apparatus in the
Euglenozoa is remarkably similar, consisting of two parallel basal
bodies and three asymmetrically distributed microtubular roots.
Many taxa have a microtubular corset that supports the cell surface. Previous studies of euglenozoan ultrastructure named the
three microtubular roots according to their position in the cell; for
example, the microtubular roots on the dorsal side, ventral side and
middle of the cell were called the “dorsal root”, “ventral root” and
“intermediate root”, respectively. It is more consistent to instead
apply the universal terminology used for the eukaryotic flagellar
apparatus to the euglenozoan root system as recommended by
Yubuki et al. [71]: the “intermediate root” is R1, the “ventral root”
is R2, and the “dorsal root” is R3.
The free-living biflagellate Bodo (eubodonid) and the fish parasite Cryptobia (parabodonid) share the typical microtubular root
system of euglenozoans [12,42] (Fig. 5d and e). The R2 microtubules
in kinetoplastids support the feeding apparatus (if present) and
form a bundle called the “reinforced microtubular band” (MTR).
The parasitic trypanosomatids have a significantly reduced flagellar apparatus consisting of only one flagellum stemming from the
anterior B2. The highly reduced posterior B1 is called the “protobasal body”. All of the cytoskeletal elements, which are normally
associated with the posterior B1 are absent in trypanosomatids.
Like in other eukaryotes, R3 is associated with B2 in trypanosomatids, but it forms a bundle of only four microtubules, called the
“microtubule quartet” (MTQ), which is a key feature of trypanosomatid cells and the only remnant of R3 [19,30] (Fig. 5f). T. brucei, in
particular, was shown to have a distinctive hairpin-shaped “bilobe”
structure that is associated with the MTQ (R3), contains a specific
isoform of centrin (TbMORN) and wraps around the flagellar pocket
[19,38]. A similar cytoskeletal element, called the “pellicle microtubule organizing center” (pMTOC), was inferred to play a critical
role in the organization and inheritance of the complex system of
pellicle strips in euglenids [67], which are close relatives to kinetoplastids. It is possible that the proposed pMTOC is homologous with
the bilobe structure in trypanosomatids and was therefore present
in the most recent ancestor of all euglenozoans. This hypothesis can
be tested by showing that the same isoform of centrin, found in the
bilobe structure of T. brucei, is also present in a similar structure in
euglenids (i.e., a pMTOC).
9. Independent streamlining and complexity in parasitic
protists
Major modifications of the microtubule root system in parasitic
protists reflect adaptations associated with different mode of motility, attachment and nutrition within different host compartments.
On one hand, some parasitic protists have independently evolved
complex structures to survive within different host environments
(e.g., the ventral disc in Giardia, the rostellum in Oxymonas, and the
apical complex in Toxoplasma). On the other hand, the evolutionary
transition from free-living lifestyles to parasitic lifestyles in different lineages of protists has involved the progressive reduction of
the ancestral feeding apparatus and associated microtubular roots.
Typical traits found in free-living predatory flagellates, such as a
split R2 that reinforces a ventral feeding groove, is generally lost
in parasitic lineages (except retortamonads); however, remnants
of the R2 remain in many parasitic lineages such as in oomycetes,
diplomonads and kinetoplastids. Free-living protists usually have
an R3 that functions as the organizing center (MTOC) for the
microtubules that support the dorsal surface of the cell, called a
“corset”, a “dorsal fan” or “cytoplasmic microtubules”. Many par-
7
asitic protists have lost R3 but still retain an array of cytoplasmic
microtubules under the cell surface (e.g., kinetoplastids, opalinids
and retortamonads). Therefore, a general trend in the evolution of
the microtubular cytoskeleton in different groups of parasitic protists is the independent loss of traits (i.e., streamlining) present in
their free-living ancestors.
Free-living, kinetoplastids, for instance, have three microtubular
roots (R1, R2 and R3) and two flagella associated with two parallel basal bodies (B1 and B2), while trypanosomatids only have
R3 and a single anterior directed flagellum associated with one
mature basal body (B2). Although the zoospores of oomycetes have
a flagellar apparatus that is similar to free-living stramenopiles
(e.g., bicosoecids), symbiotic slopalinids and the parasitic Blastocystis have lost the stramonopile-type microtubular root system
altogether. Moreover, aside from some reduced microgametes, apicomplexans have essentially lost the flagellar apparatus from their
free-living (myzocytosis-feeding) ancestors.
By contrast, endobiotic retortamonads (e.g., Retortamonas) and
some parasitic diplomonads (e.g., Hexamita and Spironucleus) have
retained the excavate-type microtubular root system found in their
free-living ancestors. Other parasitic diplomonads (e.g., Giardia)
have substantially increased the complexity of microtubular structures with new innovations that facilitate their parasitic lifestyles.
This added complexity is built upon homologs of the ancestral
roots (e.g., R2 and R3) [6,58], but genomic data suggest that certain traits (e.g., the ventral disc in Giardia) are built from proteins
that do not have homologs with known cytoskeletal proteins [22].
Other very different lineages of parasites, such as hypermastigid
parabasalids, oxymonads and opalinids, thrive within animal guts
and have increased cytoskeletal complexity by multiplying a flagellar mastigont system. These lineages have much larger cells than
their free-living ancestors and have up to thousands of flagella that
generate currents to bring nutrients across the surface of their cells.
The most recent common ancestor of eukaryotes had a complex (excavate-type) flagellar apparatus that was independently
streamlined in several different ways across the tree of eukaryotes
[68]. This general pattern of streamlining is consistent with the
independent evolutionary transitions to parasitic lifestyles from
free-living ancestors representing very distantly related groups of
eukaryotes. These patterns of ultrastrutural streamlining are also
consistent with the reoccurring loss of functional redundancy in the
genome of several different groups of parasitic organisms, such as in
microsporidians, Ichthyophthirius, Helicosporidium and Mycoplasma
[17,21,25,49,64]. Nonetheless, detailed comparisons of the flagellar apparatus in microbial eukaryotes remain difficult because the
number of organisms that have been adequately described at the
ultrastructural level is relatively small compared to the total number of known species. Improvements in our overall understanding
of evolutionary parasitology will depend on continued efforts to
discover and characterize both parasitic species and their nearest
free-living relatives using both culture-dependent and cultureindependent methods.
Acknowledgements
This work was supported by the Tula Foundation’s Center
for Microbial Diversity and Evolution at the University of British
Columbia. NY is also supported by the project BIOCEV (Biotechnology and Biomedicine Centre of the Academy of Sciences and Charles
University) (CZ.1.05/1.1.00/02.0109) from the European Regional
Development. IČ was supported by the Czech Science Foundation
(project GA14-14105S). BSL was also supported by the Canadian
Institute for Advanced Research, Program in Integrated Microbial
Biodiversity.
Please cite this article in press as: N. Yubuki, et al., Evolution of the microtubular cytoskeleton (flagellar apparatus) in parasitic protists,
Mol Biochem Parasitol (2016), http://dx.doi.org/10.1016/j.molbiopara.2016.02.002
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8
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Please cite this article in press as: N. Yubuki, et al., Evolution of the microtubular cytoskeleton (flagellar apparatus) in parasitic protists,
Mol Biochem Parasitol (2016), http://dx.doi.org/10.1016/j.molbiopara.2016.02.002